Weanling male rats were fed a riboflavin deficient diet for 5 weeks and enlargement of the liver mitochondria and discontinuity of the outer membrane were observed. The content of phospholipids was slightly increased in the deficient mitochondria, and decrease in phosphatidylcholine and increase in phosphatidylethanolamine were shown respectively. In comparison with the mitochondria, the content and distribution of phospholipids in microsomes were not affected. The fatty acid composition of phosphatidylcholine in mitochondria and microsomes was remarkably altered by the deficiency, and increases in palmitic and linoleic acids and decrease in arachidonic acid were demonstrated. The incorporation of 32P into diphosphatidylglycerol in mitochondria was reduced by the deficiency. The incorporation into other phospholipids was not significantly altered, whereas the incorporation into the subspecies of phosphatidylcholine was variously affected. By the intraperitoneal injection of riboflavin to the deficient rats, normalization of the mitochondrial size and fatty acid composition of liver mitochondrial lipids was observed. However, decreased incorporation of 32P into diphosphatidylglycerol in mitochondria was not recovered COMPLETELY AT 40 Hours after the injection, and in the mitochondrial lipids linoleic acid was higher and arachidonic acid was lower than respective controls at 60 hours.